This invention relates to the chemical synthesis of 2'-O-methyl, 3'-O-methyl and 5'-O-methyl nucleosides.
The following is a brief description of synthesis of methoxy nucleosides. This summary is not meant to be complete but is provided only for understanding of the invention that follows. This summary is not an admission that all of the work described below is prior art to the claimed invention.
Sugar modifications, such as 2'-O-methyl, have been discovered in a variety of naturally occurring RNA (e.g., tRNA, mRNA, rRNA; reviewed by Hall, 1971 The Modified Nucleosides in Nucleic Acids, Columbia University Press, New York; Limbach et al., 1994 Nucleic Acids Res. 22, 2183). In an attempt to understand the biological significance, structural and thermodynamic properties, and nuclease resistance of these sugar modifications in nucleic acids, several investigators have chemically synthesized nucleosides, nucleotides and phosphoramidites containing various sugar modifications and incorporated them into oligonucleotides. There are several reports in the literature describing the synthesis of 2'-O-methyl nucleosides, 2'-O-methyl nucleotides, 2'-O-methyl phosphoramidites and oligonucleotides containing 2'-O-methyl substitutions (Broom and Robins, 1965 J. Am. Chem. Soc. 87, 1145; Martin et al., 1968 Biochemistry, 7, 1406; Robins et al., 1974 J. Org. Chem. 39, 1891; Inoue et al., 1987 Nucleic Acids Res. 15, 6131; Cotten et al., 1991 Nucleic Acids Res. 19, 2629; Andrews et al., 1994 J. Heterocyclic Chem. 31, 765; Beigelman et al., 1995 Nucleosides & Nucleotides 14, 421; Sproat et al., 1990 Nucleic Acids Res. 18, 41).
Broom and Robins, 1965 J. Am. Chem. Soc. 87, 1145 and Martin et al., 1968 Biochemistry, 7, 1406, describe the synthesis of 2'-O-methyl ribonucleotides involving mono-methylation of a 2',3'-cis-diol system of a ribonucleoside with diazomethane. This procedure gives rise to a mixture of 2'- and 3'-O-methyl nucleosides in 20-40% combined yield. The two isomers are then separated by ion-exchange chromatography.
Robins et al., 1974 J. Org. Chem. 39, 1891, describe the treatment of a methanolic solution of uridine with diazomethane (in glyme) in the presence of stannous chloride dihydrate (in methanol) to synthesize 2'-O-methyluridine (58% yield). This reaction also yielded a significant fraction (28%) of 3'-O-methyluridine which is purified away from the 2'-O-methyl form by chromatography.
Inoue, Japanese Patent Publication No. 61291595 and Inoue et al., 1987 Nucleic Acids Res. 15, 613, describe a process for the synthesis of 2'-O-methyl ribonucleosides involving alkylation of 3',5'-O-(tetraisopropyldisiloxane-1,3-diyl) (TIPDS)-ribonucleosides with methyl iodide. Inoue et al., state that (page 6133, second main paragraph):
"Treatment of 3',5'-O-TIDPS-uridine (1) with benzoyl chloride . . . in N,N-dimethylacetamide in the presence of triethylamine . . . selectively gave the N.sup.3 -benzoylated derivative (2) in 70.5% yield. Then, 2 was treated with CH.sub.3 I . . . in benzene in the presence of Ag.sub.2 O . . . at 40.degree. C. overnight to give the N.sup.3 -benzoyl-2'-O-methyl derivative (3, 84.5%). Debenzoylation of 3 with dil. NH.sub.4 OH followed by removal of TIPDS group with 0.5N HCl afforded 2'-O-methyluridine . . . in 84% yield." PA1 "Trichloroacetimidate-2-O-alkyl-3,5-O-TIPS-.beta.-D-ribofuranoside glycosylates silylated nucleobases in a fast high-yielding and stereoselective reaction promoted by trimethylsilyl trifluoromethanesulfonate. This method has been applied to the synthesis of 2'-O-alkyl ribonucleosides further transformed to building blocks ready for oligo(2'-O-alkyl)ribonucleotide construction." PA1 "Retrosynthetic analysis showed that 3-O-alkylated derivatives of 1,2:5,6-di-O-isopropylidene(IP)-.alpha.-D-allofuranose (1) could be transformed to the related 2'-O-alkyl ribofuranosides by selective degradation of the C1-C2 bond with subsequent cyclization of the generated C2-formyl group to the C5--OH." (Page 421, third paragraph) PA1 "Among different methods of indirect introduction of a methyl group, the use of 1-alkylthioalkyl intermediates seems to be the most promising. Although methods of synthesis of methylthiomethyl ethers of nucleosides and carbohydrates are well developed, their transformation into a methyl group sometimes requires additional steps. We were interested in the testing of more reactive methylthiophenyl ethers as precursors for methyl ethers. We found that methylthiophenyl ethers could be smoothly introduced by treating appropriately protected nucleosides or carbohydrates with PhSMe/Bz.sub.2 O.sub.2 in the presence of DMAP. Nucleoside 19 afforded methythiophenyl ether 20 in 65-70% yield, and .alpha.-ribofuranose 21 was transformed into .beta.-furanose 22 in 60% yield. Different attempts to radically (Bu.sub.3 SnH, Bz.sub.2 O.sub.2) reduce the thiophenyl group of furanose 22 were not successful, providing only starting material. However, under the same conditions, nucleoside 20 afforded 2'-O-Me derivative 24 in 70% yield.
Srivastava and Roy, U.S. Pat. No. 5,214,135, describe the synthesis of 2'-O-methyl nucleosides using an approach similar to Inoue et al., supra, except that the reaction with methyl iodide/silver oxide was carried out at 25.degree. C. for 24-46 hr with an 80-86% yield. This reaction, similar to the one described by Inoue et al., supra, also gave rise to the 3'-O-methyl isomer in 6-8% yield.
Parmentier et al., 1994 Tetrahedron 50, 5361, describe a convergent synthesis of 2'-O-methyl uridine. This procedure uses a multi-step process involving--"a facile obtention of the 2'-O-methyl sugar synthon using totally selective and efficient methylation conditions; . . . a stereoselective high-yield condensation with an uracil derivative, yielding the desired .beta.-form with a satisfactory anomeric excess." (page 5361, fifth paragraph).
Chanteloup and Thuong, 1994 Tetrahedron Letters 35, 877, describe synthesis of 2'-O-alkyl ribonucleosides using trichloroacetimidate D-ribofuranosides as ribosyl donors. They state in the abstract on page 877--
Beigelman et al., 1995 Nucleosides & Nucleotides 14, 421, describe three different approaches to the synthesis of 2'-O-methyl nucleosides. They state that--
Method 1:
Method 2:
"The 3'-O-TBDMS-derivatives of protected ribonucleosides are byproducts obtained during the preparation of 2'-O-TBDMS derivatives--key building blocks in oligoribonucleotide synthesis. At the same time, 3'-O-TBDMS-isomers could be useful starting compounds in the preparation of 2'-O-methyl-3'-O-phosphoramidites. We explored this possibility on cytidine derivative 14. Reaction of 3'-O-TBDMS-5'-O-DMT-N.sup.4 -i-Bu-cytidine (14) with Ag.sub.2 O--CH.sub.3 I using a modified method of Ohtsuka et al. (supra) yielded 3'-O-TBDMS-5'-O-DMT-N.sup.4 -i-Bu-2'-O-methyl cytidine (15) in 26% yield. The 2'-O-TBDMS isomer 16 was also obtained (22% yield) along with the starting 3'-O-isomer (18%). When 2'-O-TBDMS-5'-O-DMT-N.sup.4 -i-Bu-cytidine (16) was subjected to the same reaction conditions, the same mixture of products was obtained. These results show that under the above reaction conditions migration of the TBDMS group accompanies the methylation reaction and methylation takes place selectively at the 2'-OH position." (Page 422, second full paragraph)
Method 3:
Haga et al., 1972 Carbohydrate Res. 21, 440 describe a "facile route" to the synthesis of 2- and 3-O-methyl-D-ribose from 3-O-methyl-D-allose.
Nair et al., 1982, Synthesis 8, 670, describes modification of nucleic acid bases via radical intermediates.
Leonard et al., 1992, Nucleosides & Nucleotides, 11, 1201, describe a method for the preparation of protected 2'-O-methylguanosine. This procedure is distinct from the one described in the instant invention.
Wagner et al., 1991, Nucleic Acids Res., 19, 5965, describes a method for alkylation of ribonucleosides.
The information disclosed in the references cited above are distinct from the presently claimed invention since they do not disclose and/or contemplate the processes for the synthesis of the methoxy nucleosides as claimed in the instant invention.